Improved process for the enantioselective hydrolysis of prochiral diethyl malonates catalyzed by pig liver esterase

Article abstract of DOI:10.1055/s-2005-871548

An improved process for the enantioselective hydrolysis of prochiral 2-aryl-2-alkyl-disubstituted diethyl malonates catalyzed by pig liver esterase (PLE) was developed. With diethyl 2-phenyl-2-methylmalonate as a model substrate, the highest enantioselect

Full text of DOI:10.1055/s-2005-871548

1746
LETTER
Improved Process for the Enantioselective Hydrolysis of Prochiral Diethyl
Malonates Catalyzed by Pig Liver Esterase
I
mprove
d
Proc
a
ess
f
or the
b
Enantioselec
l
tive
E
n
o
zymatic
H
ydrolysi
D
s
of Malonates omínguez de María,a Beate Kossmann,b Nicole Potgrave,b Stefan Buchholz,c Harald Trauthwein,c
Oliver May,a Harald Gröger*a
a
Degussa AG, Service Center Biocatalysis, Rodenbacher Chaussee 4, 63457 Hanau-Wolfgang, Germany
b
Degussa AG, Creavis Technologies & Innovation, Paul-Baumann-Straße 1, 45764 Marl, Germany
c
Degussa AG, Project House Proferm, Rodenbacher Chaussee 4, 63457 Hanau-Wolfgang, Germany
Fax +49(6181)592961; E-mail: harald.groeger@degussa.com
Received 22 February 2005
CO2Et
CO2Et
CO2H
R2
R1
R2
R1
pig liver esterase
Abstract: An improved process for the enantioselective hydrolysis
of prochiral 2-aryl-2-alkyl-disubstituted diethyl malonates cata-
lyzed by pig liver esterase (PLE) was developed. With diethyl 2-
phenyl-2-methylmalonate as a model substrate, the highest enanti-
oselectivities (96% ee; R-isomer) were achieved when carrying out
the process in the presence of a suitable mixture of cosolvents
(buffer–i-PrOH–t-BuOH, 8:1:1). The process also works efficiently
at higher substrate concentrations. The reaction was carried out on
a multi-gram preparative scale, leading to a conversion of >95%, an
enantioselectivity of 96% ee, and a yield of 83% at a substrate con-
centration of 200 mM.
suitable
solvent media
CO2Et
1
(R)-2
1a: R1 = Ph, R2 = CH3,
1b: R1 = Ph, R2 = allyl
Scheme 1 General procedure for the enzymatic hydrolysis of
prochiral diester malonates
shown a moderate to high enantioselectivity of up to 80–
85% ee [(R)-2a enantiomer];8,9 the principle of this syn-
thesis is shown in Scheme 1.
Key words: asymmetric synthesis, enzymes, hydrolyses, mal-
onates, solvent effects
Previously, the improvement of the enantioselectivity of
esterases by addition of small amounts of different cosol-
vents had been described in several papers.1,10–14 Ethanol
has been reported as the best cosolvent for the hydrolysis
of diethyl 2-(4-methoxyphenyl)-2-methyl malonate.1
However, upon the addition of ethanol (20%) the enantio-
selectivitiy in the formation of (R)-2a increased only from
81% to 90% ee (Table 1, entries 1, 3; 20 mM substrate
The importance of optically active malonate monoesters
as key pharmaceutical intermediates is well known.1–4
The enzymatic approach by hydrolyzing prochiral dialkyl
malonates 1 (Scheme 1) in the presence of pig liver es-
terase (PLE) is an attractive route for producing chiral
building blocks, since high conversions can be obtained
under mild reaction conditions and easy work-up is possi-
ble.5 Enantioselectivity varies dependent on the substrate
pattern. However, despite the potential of this PLE-cata-
lyzed desymmetrization process for large scale applica-
tions, there are only a few studies regarding process
development.1,3,4,6 The particular challenges are still sim-
ple and economically attractive processes based on a
small amount of enzymes and high substrate concentra-
tions resulting in both high conversions (>90%) and enan-
tioselectivities (>95%). The present paper addresses these
issues and demonstrates suitability of the developed im-
proved process for preparative (multi-gram) scale applica-
tions.
Table 1 Influence of Cosolvents and Mixtures thereof in the Enzy-
matic Hydrolysis of 1aa
PLEb
Cosolvent (%) Conversionc (time) ee (%)
1
2
3
4
5
6
7
8
50 mg
30 mg
50 mg
30 mg
30 mg
30 mg
300 mLd
40 mg
–
>90% (3 h)
90% (10 h)
>90% (10 h)
16% (12 h)
3% (12 h)
81
MeOH (20)
EtOH (20)
PrOH (20)
BuOH (20)
i-PrOH (20)
t-BuOH (20)
87
90
n.d.e
n.d.e
94
>90% (3 h)
>90% (3 h)
>90% (2 h)
As a substrate for the initial optimization study diethyl 2-
phenyl-2-methyl malonate (1a) was chosen. Commercial
PLE, which is a crude preparation consisting of several
isoenzymes, was used as a biocatalyst. Notably, cloning
and expression of a distinct isoenzyme in Pichia pastoris
was achieved by the Bornscheuer group recently.7 Ac-
cording to the literature, the PLE-catalyzed hydrolysis has
95
i-PrOH (10)
t-BuOH (10)
96
a Phosphate buffer (50 mM, pH 7.0), cosolvent, substrate (20 mM),
30 °C, volume (50 mL) [entry 1: non-optimized reaction time].
b Commercial PLE from Sigma (ca. 40 U/mg).
c Conversion measured as consumption of NaOH.
d Liquid PLE-preparation (300 mL; Eupergit-immobilized PLE) from
Röhm was used.
SYNLETT 2005, No. 11, pp 1746–1748
0
7
.0
7
.2
0
0
5
Advanced online publication: 14.06.2005
DOI: 10.1055/s-2005-871548; Art ID: G04405ST
© Georg Thieme Verlag Stuttgart · New York
e Not determined.

LETTER
Improved Process for the Enantioselective Enzymatic Hydrolysis of Malonates
1747
concentration). Furthermore, upon addition of methanol
(20%) less satisfactory enantioselectivity was observed
(Table 1, entry 2), and addition of propanol and butanol
(20%) led to very low conversions, 16% and 3%, respec-
tively (Table 1, entries 4, 5).
Table 2 Enzymatic Hydrolysis of 1a at Various Substrate Concen-
trationsa
Substrate concn Time
Conversion Relative
ee
(mM)
(h)
productivity (%)b (%)
1
2
3
4
5
6
20
2
>95%
>95%
>95%
>95%
>95%
>95%
100
100
100
78
96
96
96
96
96
95
Non-alcohol solvents like acetone and acetonitrile low-
ered the enantioselectivity and conversion of the reaction
(results not shown). However, we were pleased to find
that the presence of branched alcohols not only gave high
conversions but also significantly enhanced enantio-
selectivities. For example, the employment of isopropanol
(20%) as an additive increased the enantioselectivity up to
94% ee, accompanied by a high conversion of >90%
(Table 1, entry 6). The use of tert-butanol, which had been
previously described for other molecules than malonates
with PLE and other hydrolases,11,12 also resulted in high
enantioselectivity (95% ee; Table 1, entry 7).
50
5
100
200
250
300
10
25.5
38
68
66
44
a Phosphate buffer (80%, 50 mM, pH 7), i-PrOH (10%), t-BuOH
(10%), PLE (40 mg), 30 °C, 50 mL volume.
b Relative to the productivity at 20 mM substrate concentration (The
productivity was measured in mmol of product formed per h and mg
of PLE).
A further improvement was achieved in the presence of
mixtures of cosolvents. When a mixture of buffer/isopro-
panol/tert-butanol (8:1:1) was applied, the enantioselec-
tivity was slightly better with 96% ee and conversion
remained high (>90%; Table 1, entry 8). Thus, the use of
(mixtures of) cosolvents for the PLE-catalyzed synthesis
of (R)-2a represents both a simple and efficient means of
improving the enantioselectivity (from 80–85 to 96% ee).
In addition, high conversions are also obtained in the pres-
ence of cosolvents, albeit reaction rates themselves are
lower than those in pure aqueous buffer (kinetic data not
shown). A pH of six to seven and a reaction temperature
of 30–35 °C were found to be optimal with respect to both
conversion and enantioselectivity.
pendent of the substrate concentration (range of 20–300
mM), high conversions of >95% were achieved (Table 2,
entries 1–6). Also the enantioselectivity remained high at
95–96% ee. However, whereas productivity remains sat-
isfactory at substrate concentrations of up to 200–250
mM, the productivity decreased to 44% when the sub-
strate concentration was increased further to 300 mM.
Thus, a subsequent multi-gram scale synthesis of the op-
tically active ethyl malonate (R)-2a (300 mL scale) was
carried out at a substrate concentration of 200 mM
(Scheme 3). The reaction proceeds comparable to the re-
action on a 50 mL scale, and gave a conversion of >95%
and an enantioselectivity of 96% ee. After extractive
work-up, which turned out to be suitable for this reaction
mixture, 11.2 g of the desired product were isolated corre-
sponding to 83% yield.
CO2Et
CO2Et
CO2Et
CO2H
pig liver esterase
Ph
buffer,
with/without cosolvent
Ph
(R)-2b
1b
conversion
(%)
ee
(%)
pig liver esterase
cosolvent [%(v/v)]
CO2Et
CO2Et
CO2Et
CO2H
(240 mg)
H3C
Ph
H3C
Ph
>90
>90
49
87
without cosolvent
t-BuOH (10), i-PrOH (10)
buffer/i-PrOH/t-BuOH
(8:1:1),
pH 7.0, 30 °C, 25 h
(R)-2a
1a
>95% conversion,
83% yield (11.2 g)
96% ee
Scheme 2 PLE-catalyzed hydrolysis of 1b with and without cosol-
vents (40 mM of substrate, 50 mM phosphate buffer, pH 7.0, 30 °C,
40 mg PLE, 50 mL volume, reaction mixtures were stirred overnight;
absolute configuration was assigned according to analogous molecu-
les).15
200 mM substrate conc.
(50 g/L),
300 mL scale
Scheme 3 Enantioselective hydrolysis of 2-methyl-2-phenyl-malo-
nate, 1a, on a multi-gram scale.
The optimized reaction media also turned out to be suit-
able for the application of other types of disubstituted ma-
lonates. For example, an enhancement of the
enantioselectivity from 49% to 87% ee was observed for
the hydrolysis of diethyl 2-phenyl-2-allylmalonate (1b) as
a substrate (Scheme 2).
In conclusion, an improved process for PLE-catalyzed
enantioselective hydrolysis of prochiral dialkyl malonates
has been realized, which allows the preparation of the tar-
get products at high substrate concentrations of, e.g., 200
mM with both high conversions (up to >95%) and enanti-
oselectivities (up to 96% ee) at reasonable reaction times.
Feasibility of the process for multi-gram synthesis has
been demonstrated. A key feature of this process is the
designed reaction media, consisting of a mixture of cosol-
vents, which ensures a high enantioselectivity. Expanding
the application range of this process is in progress.
The best conditions for hydrolysis were selected, and next
we increased the substrate concentration, since sufficient
substrate concentration is a prerequisite for large scale
applicability; the results are depicted in Table 2. Inde-
Synlett 2005, No. 11, 1746–1748 © Thieme Stuttgart · New York

1748
P. D. de María et al.
LETTER
Pig liver esterase (PLE) was purchased from Sigma-Aldrich. All re-
agents are commercially available and were used without further
purification. The synthesis of the substrates diethyl 2-phenyl-2-
methyl malonate (1a), and diethyl 2-phenyl-2-allyl malonate (1b),
as well as the racemates rac-2, was done according to standard
operations.8 The enantioselectivity was determined by HPLC-anal-
ysis using a chiral column OD. The separation of enantiomer peaks
were achieved with a flow of 0.25 mL/min at 240 nm, by using a
mobile phase composed of hexane–i-PrOH–trifluoroacetic acid
(93:7:1.5).
References
(1) Patel, R.; Banerjee, A.; Chu, L.; Brozozowski, D.; Nanduri,
V.; Szarka, L. J. J. Am. Oil Chem. Soc. 1998, 75, 1473.
(2) Fadel, A.; Arzel, P. Tetrahedron: Asymmetry 1995, 6, 893.
(3) Fadel, A.; Garcia-Argote, S. Tetrahedron: Asymmetry 1996,
7, 1159.
(4) Fadel, A.; Vandromme, L. Tetrahedron: Asymmetry 1999,
10, 1153.
(5) Drauz, K.; Waldmann, H. Enzyme Catalysis in Organic
Synthesis, 2nd Ed., Vol.; Wiley-VCH: Weinheim, 2002,
Chap. 11.
(6) Klotz-Berendes, B.; Kleemiss, W.; Jegelka, U.; Schäfer, H.
J.; Kotila, S. Tetrahedron: Asymmetry 1997, 8, 1821.
(7) Musidlowska, A.; Lange, S.; Bornscheuer, U. T. Angew.
Chem. Int. Ed. 2001, 40, 2851; interestingly, remarkable
differences in enantioselectivities between commercially
available crude PLE and the recombinant PLE-isoenzyme
are reported therein.
(8) Toone, E.; Jones, B. Tetrahedron: Asymmetry 1991, 2, 1041.
(9) Schneider, M.; Engel, N.; Boensmann, H. Angew. Chem.,
Int. Ed. Engl. 1984, 23, 64; Angew. Chem. 1984, 96, 54.
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Tetrahedron Lett. 1985, 26, 4957.
Enzymatic hydrolysis was carried out in a Titrino reaction apparatus
with a total volume of 50 mL. The aqueous media consists of a 50
mM phosphate buffer, and cosolvent(s) was/were added. The sub-
strate concentration depended on the experiment (20–300 mM), as
well as the amount of enzyme used. The hydrolysis was controlled
by adjusting the pH to 7.0 by addition of NaOH (0.25–1 N). The re-
action was stopped after consumption of one equivalent of NaOH.
The aqueous reaction mixture was extracted with two aliquots of
CH2Cl2 (2 × 50 mL). Afterwards, the aqueous phase was acidified
with HCl (1 M) till pH < 2.5, and then re-extracted with four ali-
quots of CH2Cl2 (4 × 50 mL). The organic phase was dried with
MgSO4, filtered, and concentrated over reduced pressure, yielding
the optically active products (R)-2 (HPLC and NMR analysis).
Analytical data (NMR): Ethyl 2-phenyl-2-methyl-ethylmalonate
[(R)-2a]: 1H NMR: d = 1.25 (3 H, t), 1.9 (3 H, s), 4.25 (2 H, m), 7.35
(5 H, Ar). Ethyl 2-phenyl-2-allyl-ethylmalonate [(R)-2b]: 1H NMR:
d = 1.25 (3 H, t), 3.05 (1 H, m), 3.2 (1 H, m), 4.2 (2 H, m), 5.1 (2 H,
dd), 5,75 (1 H, m), 7.35 (5 H, Ar).
(11) Guanti, G.; Banfi, L.; Narisano, E.; Riva, R.; Thea, S.
Tetrahedron Lett. 1986, 27, 4639.
(12) Guanti, G.; Banfi, L.; Narisano, E. Tetrahedron Lett. 1989,
30, 2697.
(13) Lam, L. K. P.; Raymond, A. H. F.; Jones, J. B. J. Org. Chem.
1986, 51, 2047.
(14) Chartrain, M.; Maligres, P.; Cohen, D.; Upadhyay, V.;
Pecore, V.; Askin, D.; Greashan, R. J. Biosci. Bioeng. 1999,
87, 386.
(15) Arzel, P.; Freida, V.; Weber, P.; Fadel, A. Tetrahedron:
Asymmetry 1999, 10, 3877.
Acknowledgment
Dr. Pablo Domínguez de María is grateful for an EU-Marie Curie
Postdoctoral Industrial Fellowship. The authors thank Dr. Stefan
Menzler (Degussa Specialty Polymers, Röhm GmbH and Co. KG)
for providing us with samples of Eupergit-immobilized PLE.
Synlett 2005, No. 11, 1746–1748 © Thieme Stuttgart · New York